Brigitte Müller-Hilke

Plasma cell differentiation in T-independent type 2 immune responses is independent of CD11chigh dendritic cells

Dendritic cells (DC) play an important role as antigen‐presenting cells in T cell stimulation. Interestingly, a number of recent studies also imply DC as critical accessory cells in B cell activation, isotype switching and plasma blast maintenance. Here we use the conditional in vivo ablation of CD11chigh DC to investigate the role of these cells in T‐independent typeu20042 immune responses. We show that CD11chigh DC are dispensable for the initiation and maintenance of a primary immune response against the T‐independent typeu20042 antigen (4‐hydroxy‐3‐nirophenyl)acetyl‐Ficoll. Our results suggest that support for plasma cell formation in T cell‐independent immune responses can be provided by non‐DC such as stromal cells, or is independent of external signals. Interestingly, we found plasma blasts to express CD11c and to be diphtheria toxin‐sensitive in CD11c‐diphtheria toxin receptor‐transgenic mice, providing a unique tool for future analysis of in vivo aspects of plasma cell biology.

Immunomodulatory effects of holothurian triterpene glycosides on mammalian splenocytes determined by mass spectrometric proteome analysis

Spleen is a prime organ in which immuno-stimulation takes place in mammalians. Proteome analysis was used to investigate the elicited effects on mouse splenocytes upon exposure to holothurian triterpene glycosides. Cucumarioside A(2)-2, and Frondoside A, respectively, have been used to in-vitro stimulate primary splenocyte cultures. Differential protein expression was monitored by 2D gel analysis and proteins in spots of interest were identified by MALDI ToF MS and nano LC-ESI Q-ToF MS/MS, respectively. Differential image analysis of gels from control vs. gels from stimulated primary splenocyte cultures showed that approximately thirty protein spots were differentially expressed. Prime examples of differentially expressed proteins are NSFL1 cofactor p47 and hnRNP K (down-regulated), as well as Septin-2, NADH dehydrogenase [ubiquinone] iron-sulfur protein 3, and GRB2-related adaptor protein 2 (up-regulated). Immuno-analytical assays confirmed differential protein expression. Together with results from proliferation and cell adhesion assays, our results show that cellular proliferation is stimulated by holothurian triterpene glycosides. In conclusion, holothurian triterpene glycosides are thought to express their immuno-stimulatory effects by enhancing the natural cellular defense barrier that is necessary to fight pathogens and for which lymphocytes and splenocytes have to be recruited constantly due to limited lifetimes of B-cells and T-cells in the body.

Senescence determines the fate of activated rat pancreatic stellate cells

In chronic pancreatitis (CP), persistent activation of pancreatic stellate cells (PSC) converts wound healing into a pathological process resulting in organ fibrosis. Here, we have analysed senescence as a novel mechanism involved in the termination of PSC activation and tissue repair. PSC senescence was first studied in vitro by establishing long‐term cultures and by applying chemical triggers, using senescence‐associated β‐Galactosidase (SA β‐Gal) as a surrogate marker. Subsequently, susceptibility of PSC to immune cell‐mediated cytolysis was investigated employing cocultures. Using the model of dibutyltin dichloride‐induced CP in rats, appearance of senescent cells was monitored by immunohistochemistry and immunofluorescence, and correlated with the progression of tissue damage and repair, immune cell infiltration and fibrosis. The results indicated that long‐term culture and exposure of PSC to stressors (doxorubicin, H2O2 and staurosporine) induced senescence. Senescent PSC highly expressed CDKN1A/p21, mdm2 and interleukin (IL)‐6, but displayed low levels of α‐smooth muscle actin. Senescence increased the susceptibility of PSC to cytolysis. In CP, the number of senescent cells correlated with the severity of inflammation and the extension of fibrosis. Areas staining positive for SA β‐Gal overlapped with regions of fibrosis and dense infiltrates of immune cells. Furthermore, a close physical proximity of immune cells and activated PSC was observed. We conclude that inflammation, PSC activation and cellular senescence are timely coupled processes which take place in the same microenvironment of the inflamed pancreas. Lymphocytes may play a dual‐specific role in pancreatic fibrogenesis, triggering both the initiation of wound healing by activating PSC, and its completion by killing senescent stellate cells.

Trefoil factor 3 is induced during degenerative and inflammatory joint disease, activates matrix metalloproteinases, and enhances apoptosis of articular cartilage chondrocytes

OBJECTIVEnTrefoil factor 3 (TFF3, also known as intestinal trefoil factor) is a member of a family of protease-resistant peptides containing a highly conserved motif with 6 cysteine residues. Recent studies have shown that TFF3 is expressed in injured cornea, where it plays a role in corneal wound healing, but not in healthy cornea. Since cartilage and cornea have similar matrix properties, we undertook the present study to investigate whether TFF3 could induce anabolic functions in diseased articular cartilage.nnnMETHODSnWe used reverse transcriptase-polymerase chain reaction, Western blot analysis, and immunohistochemistry to measure the expression of TFF3 in healthy articular cartilage, osteoarthritis (OA)-affected articular cartilage, and septic arthritis-affected articular cartilage and to assess the effects of cytokines, bacterial products, and bacterial supernatants on TFF3 production. The effects of TFF3 on matrix metalloproteinase (MMP) production were measured by enzyme-linked immunosorbent assay, and effects on chondrocyte apoptosis were studied by caspase assay and annexin V assay.nnnRESULTSnTrefoil factors were not expressed in healthy human articular cartilage, but expression of TFF3 was highly up-regulated in the cartilage of patients with OA. These findings were confirmed in animal models of OA and septic arthritis, as well as in tumor necrosis factor alpha- and interleukin-1beta-treated primary human articular chondrocytes, revealing induction of Tff3/TFF3 under inflammatory conditions. Application of the recombinant TFF3 protein to cultured chondrocytes resulted in increased production of cartilage-degrading MMPs and increased chondrocyte apoptosis.nnnCONCLUSIONnIn this study using articular cartilage as a model, we demonstrated that TFF3 supports catabolic functions in diseased articular cartilage. These findings widen our knowledge of the functional spectrum of TFF peptides and demonstrate that TFF3 is a multifunctional trefoil factor with the ability to link inflammation with tissue remodeling processes in articular cartilage. Moreover, our data suggest that TFF3 is a factor in the pathogenesis of OA and septic arthritis.

Articular cartilage-derived cells hold a strong osteogenic differentiation potential in comparison to mesenchymal stem cells in vitro

Cartilaginous matrix-degenerative diseases like osteoarthritis (OA) are characterized by gradual cartilage erosion, and also by increased presence of cells with mesenchymal stem cell (MSC) character within the affected tissues. Moreover, primary chondrocytes long since are known to de-differentiate in vitro and to be chondrogenically re-differentiable. Since both findings appear to conflict with each other, we quantitatively assessed the mesenchymal differentiation potential of OA patient cartilage-derived cells (CDC) towards the osteogenic and adipogenic lineage in vitro and compared it to that of MSC isolated from adipose tissue (adMSC) of healthy donors. We analyzed expression of MSC markers CD29, CD44, CD105, and CD166, and, following osteogenic and adipogenic induction in vitro, quantified their expression of osteogenic and adipogenic differentiation markers. Furthermore, CDC phenotype and proliferation were monitored. We found that CDC exhibit an MSC CD marker expression pattern similar to adMSC and a similar increase in proliferation rate during osteogenic differentiation. In contrast, the marked reduction of proliferation observed during adipogenic differentiation of adMSC was absent in CDC. Quantification of differentiation markers revealed a strong osteogenic differentiation potential for CDC, however almost no capacity for adipogenic differentiation. Since in the pathogenesis of OA, cartilage degeneration coincides with high bone turnover rates, the high osteogenic differentiation potential of OA patient-derived CDC may affect clinical therapeutic regimens aiming at autologous cartilage regeneration in these patients.

Reduced expression of Sfrp1 during chondrogenesis and in articular chondrocytes correlates with osteoarthritis in STR/ort mice.

To circumvent the problems of genetic and environmental diversity hampering the analysis in humans, we turned to a murine model for human knee osteoarthritis (OA) and fine mapped a previously defined OA-quantitative trait locus (QTL). We here focused on one of the candidate genes within the OA-QTL encoding the Wnt antagonist secreted frizzled related protein 1 (Sfrp1). Sequence analysis of the Sfrp1 gene in the OA strain STR/ort revealed 23 polymorphic changes with a potential to alter the gene expression. Indeed, a reduced expression in STR/ort mice was demonstrated for articular chondrocytes and hypertrophic chondrocytes of the femoral growth plate as shown by immunohistochemistry. RT-PCR of in vitro generated mesenchymal stem cells (MSC) and chondrogenically differentiated MSC (cMSC) confirmed the reduced Sfrp1 expression in STR/ort mice. This reduced Sfrp1 expression in MSC correlated with an increased amount of cytoplasmic β-catenin, a downregulation of the Wnt target gene PPARγ and an upregulation of Runx2 as well as a preferential differentiation of the MSC along the osteoblasts lineage. Given the role of Wnt signalling during chondrogenesis and in maintaining the integrity of the long lived articular chondrocytes, we conclude from our results that the reduced Sfrp1 expression in STR/ort mice not only leads to an increased activation of the Wnt/β-catenin signalling early in life but also renders the articular cartilage prone to premature ageing and to the development of OA.

HLA class II and autoimmunity: Epitope selection vs differential expression

Autoimmune diseases like rheumatoid arthritis (RA), multiple sclerosis, psoriasis and insulin-dependent diabetes mellitus are subject to a complex pathogenesis controlled by multiple genes and numerous environmental factors. The strongest genetic association is with certain HLA class II haplotypes and we here summarize the evidence supporting differential expression as a mechanism supporting the autoimmune process.

High bone mass in the STR/ort mouse results from increased bone formation and impaired bone resorption and is associated with extramedullary hematopoiesis

We here describe the novel high bone mass phenotype in STR/ort mice that leads to increased bone masses of cortical and trabecular bone and is associated with elevated osteoblast activity and impaired osteoclast function alike. Comparison of STR/ort and C57BL/6 mice reveals an increase in trabecular bone volumes of the vertebrae and at femoral metaphysis. In the females, this difference is significant as early as 2xa0months of age and at 9xa0months the females by far exceed their age matched males in all parameters measured. The increase in cortical bone mass at femoral diaphysis results from an apposition to the endosteal surface, it is significant for both sexes as early as 1xa0month of age and leads to bone marrow compression and extramedullary hematopoiesis. Altered activities of both, the osteoblast and the osteoclast contribute to the high bone mass and collectively this phenotype supports a multifactorial pathogenesis. Moreover, the spontaneous development of osteoarthritis in male STR/ort mice is suggestive of a tight correlation between trabecular bone mass and the development of degenerative changes of the articular cartilage.

Dietary Restriction-Induced Alterations in Bone Phenotype: Effects of Lifelong Versus Short-Term Caloric Restriction on Femoral and Vertebral Bone in C57BL/6 Mice

Caloric restriction (CR) is a well‐described dietary intervention that delays the onset of aging‐associated biochemical and physiological changes, thereby extending the life span of rodents. The influence of CR on metabolism, strength, and morphology of bone has been controversially discussed in literature. Thus, the present study evaluated whether lifelong CR versus short‐term late‐onset dietary intervention differentially affects the development of senile osteoporosis in C57BL/6 mice. Two different dietary regimens with 40% food restriction were performed: lifelong CR starting in 4‐week‐old mice was maintained for 4, 20, or 74 weeks. In contrast, short‐term late‐onset CR lasting a period of 12 weeks was commenced at 48 or 68 weeks of age. Control mice were fed ad libitum (AL). Bone specimens were assessed using microcomputed tomography (μCT, femur and lumbar vertebral body) and biomechanical testing (femur). Adverse effects of CR, including reduced cortical bone mineral density (Ct.BMD) and thickness (Ct.Th), were detected to some extent in senile mice (68+12w) but in particular in cortical bone of young growing mice (4+4w), associated with reduced femoral failure force (F). However, we observed a profound capacity of bone to compensate these deleterious changes of minor nutrition with increasing age presumably via reorganization of trabecular bone. Especially in lumbar vertebrae, lifelong CR lasting 20 or 74 weeks had beneficial effects on trabecular bone mineral density (Tb.BMD), bone volume fraction (BV/TV), and trabecular number (Tb.N). In parallel, lifelong CR groups showed reduced structure model index values compared to age‐matched controls indicating a transformation of vertebral trabecular bone microarchitecture toward a platelike geometry. This effect was not visible in senile mice after short‐term 12‐week CR. In summary, CR has differential effects on cortical and trabecular bone dependent on bone localization and starting age. Our study underlines that bone compartments possess a lifelong capability to cope with changing nutritional influences.

Bone resorption correlates with the frequency of CD5+ B cells in the blood of patients with rheumatoid arthritis

OBJECTIVEnThe prevention of bone resorption and subsequent joint destruction is one of the main challenges in the treatment of patients suffering from RA. Various mechanisms have previously been described that contribute to bone resorption in tightly defined cohorts. Here we analysed a cross-sectional cohort of RA patients and searched for humoral and cellular markers in the peripheral blood associated with bone resorption.nnnMETHODSnWe enrolled 61 consecutive RA patients positive for ACPA. Blood was analysed by flow cytometry to determine the percentages of regulatory T cells and B cell subpopulations. Cytokine (TNF-α, IL-6, IL-10) and ACPA levels as well as the bone resorption marker CTX-1 were determined from the patients sera. Standard clinical disease parameters were included.nnnRESULTSnMultivariate analyses showed that the percentages of CD5(+) B cells were positively correlated with CTX-1 serum levels. However, neither low-avidity ACPA nor serum IL-6 levels, both known to be produced by CD5(+) cells, were associated with CTX-1 in patients sera. There was no correlation between CTX-1 levels and clinical parameters or ACPA levels.nnnCONCLUSIONnIn summary, we found that the CD5(+) B cell population is associated with bone resorption as measured via serum CTX-1 levels in a cross-sectional cohort of RA patients. However, a possible functional link between CD5(+) B cells and bone resorption still needs to be defined.